This project has involved two separate efforts: Initially, we approached Drs. Koszalka and Krenitzky at Burroughs Wellcome and asked to provide NMR expertise to assist their work on the development of pharmacologic agents to combat AIDS. These discussions led to a collaborative effort aimed at the characterization of the active site of bacterial purine nucleoside phosphorylase, and at elucidating the basis for enzyme inhibition by the drug tubercidin (7-deazaadenosine). A series of studies was designed utilizing the transferred nuclear Overhauser Effect ("TRNOE") to characterize the enzyme-inhibitor interaction. As part of this research effort, extensive modeling of the TRNOE as a function of the NMR parameters of the enzyme-complexed and uncomplexed inhibitor and the binding/dissociation kinetics was performed, using relaxation matrix programs developed specifically for this problem.1 Since it was determined that the results depend in general on the ligand (inhibitor) exchange rate, we evaluated existing methodologies and developed several new approaches for the determination of the exchange rate. More specifically, approaches were developed involving: 1) measurements of the transverse relaxation rate for ligand nuclei using a Carr-Purcell- Meiboom-Gill (CPMG) experiment as a function of the pulse rate, and 2) measurement of T1 for the exchanging ligands as a function of the strength of the spin-lock field.2 Information related to the exchange of tubercidin with the active site, and to the structure of PNP-complexed tubercidin was derived from these studies.3 This information is important for the commercial use of this enzyme in the preparation of nucleoside drugs. A second research effort was recently initiated aimed at the development of clinically useful HIV protease inhibitors. HIV protease is a well established target enzyme for chemotherapeutic agents, but to date, no completely successful protease inhibitors have been developed and none is in clinical use. As a starting point, we will use the cyclic urea structure initially proposed by Lam and cowokers at DuPont-Merck.